Production of renewable fuel from lignocellulosic plant biomass is based on extraction of cellulosic sugars from plant cell wall material. The percentage of usable substrate (cellulose content) in plant biomass and the ease of its extraction for ethanol conversion purposes are of prime importance in efforts to improve process and cost efficiency of bioethanol production. The ease of extractability of sugar substrates is hampered by plant “recalcitrance”, a term referring to the inherent resistance of plant material to release polysaccharides and other desirable biomaterials from an interwoven matrix of desirable and undesirable materials (Lynd L R. et al., Science 251:1318-1323 (1991)). Extensive thermochemical and enzymatic treatment are needed to overcome this innate recalcitrance of plant stem biomass. Lignin content, its crosslinkages with wall carbohydrates and cell wall architecture are some known causes of recalcitrance. Furthermore, lignin processing creates inhibitory byproducts, such as phenolic and acetylated compounds, that hamper further extraction and fermentation. Phenolics and acetyl esters released during treatment of cell wall polymers can inhibit saccharification of biomass. The released acetate is also inhibitory to the organisms used to ferment the sugars into useful byproducts. Overcoming plant recalcitrance to releasing biomaterials bound in the cell wall as well as increasing the content of desirable substrates (cellulose) are therefore issues of primary importance in the development of biofuel technology.
Cellulose, composed of unbranched chains of beta-1-4 linked D-glucose units, is the most abundant biopolymer on earth and the key substrate for bioethanol production from lignocellulosic biomass. It is suggested in literature that factors such as (i) composition, number and arrangement of CESA (catalytic cellulose biosynthesis enzyme)—containing functional complexes, or rosette complexes and wall associated (KORRIGAN, KOBITO, COBRA) membrane associated (CSI) and soluble proteins (SuSy) influence the cellulose biosynthesis pathway (Endler and Persson, Arabidopsis. Mol. Plant. 2011; 4:199-211; Mizrachi et al., New Phytol. 2012; 194:54-62). Discovery of regulatory and signaling factors critical to determination of the cellulose properties (content, degree of polymerization and crystallinity) of plant cell walls and biomass are an important consideration in biomass improvement strategies (Kalluri et al. 2014, Plant Biotechnol J. 2014 December; 12(9):1207-16. doi: 10.1111/pbi.12283. Epub 2014 Nov. 3).
Lignins, complex interlinking biopolymers derived from hydroxyphenylpropanoids, provide rigidity and structure to plant cell walls for plant growth and transport of water and nutrients, and are significant contributors to plant recalcitrance. Lignins are composed primarily of syringyl (5), guaiacyl (G) and p-hydroxyphenyl (H) monolignol subunits, which are derived from sinapyl, coniferyl and p-coumaryl alcohols, respectively. The subunit ratio and resulting structure of plant lignins varies according to the genotype, environment, tissue type and maturity of the plant and as such, lignins are very heterogeneous and can vary significantly between different plants, within different tissues of a single plant and even within a single plant cell (Simmons B A et al., Curr Opin Plant Biol. 13:313-20 (2010)). This complexity and heterogeneity hinders the development of conversion technology able to process a range of sustainable feedstocks in a cost-effective manner. A lack of precise understanding of this structure has been an impediment in designing biomass improvement and biomass conversion strategies.
An increase in cellulose and a reduction of lignin content and cell wall recalcitrance, is desirable for biofuel, and pulp and paper industries. Conversely, increases in lignin content of cell walls can be desirable for production of lignin-based products such as carbon fibers. Thus, genetic manipulation of biomass feedstock to modulate cellulose and lignin biosynthesis, and sugar release efficiency hold promise both for production of improved, economically sustainable lignocellulosic biofuels (Vermerris W. et al., Crop Science 47(S3):S142-S153 (2007); Fu C. et al., PNAS 108:3803-3808 (2011)), and for creating improved cellulose- and lignin-based bioproducts.
The genus Populus represents an economically important tree crop that has been targeted for use in diverse applications from the pulp and paper industry, bioremediation, carbon sequestration and as a feedstock in the lignocellulosic biofuel industry (Dinus R J. et al., Crit. Rev. Plant Sci. 20:51-69 (2001)). Recent molecular profiling (using transcriptomics, proteomics and bioinformatics) and analysis of developmental (xylem development, [Kalluri et al., 2009]) and physiological (tension stress response) conditions under which, perennial plants such as Populus undergo enhanced cellulose biosynthesis, secondary cell wall development and biomass production has identified new genes that potentially impact biomass properties (Yang et al., 2011).
Tension wood is a special type of reaction wood formed in upper side of bending/leaning stems of woody angiosperms, and is characterized enhanced xylem (wood) cell proliferation, cellulose production in new cell wall layers [>90% cellulose], and reduced recalcitrance (Foston et al., 2001; Jung et al., 2013). Molecular profiling of tension stress response is an effective approach to understanding the biosynthetic, signaling and regulatory factors profiling underlying tension wood formation.
Recently, a study using wild Populus trichocarpa genotypes collected in the Pacific Northwest region demonstrated high phenotypic variation among the accessions in recalcitrance measured by lignin content and sugar release (Studer M H. et al., PNAS 108:6300-6305 (2011)). This study suggested that sufficient variation occurs in wild germplasm to identify specific genetic determinants of the recalcitrance trait by analysis of naturally-occurring allelic variability.
Quantitative trait loci (QTL) studies have been conducted using interspecific mapping of populations to identify genomic regions associated with cell wall phenotypes linked to recalcitrance (Novaes E. et al., New Phytologist 182:878-890 (2009); Yin T. et al., PLoS one 5:e14021 (2010)). Wegrzyn J L. et al., New Phytologist 188:515-532 (2010) demonstrated the feasibility of using linkage disequilibrium (LD)-based association mapping to validate candidate genes with putative functions in cell wall biosynthesis. The extent of LD decay in P. trichocarpa has been described by Slavov G T. et al., New Phytologist 196(3):713-25 (2012), who reported LD decay to below r2=0.2 within 2 kb in more than half of the genes, within a genomewide average 6-7 kb. Given that the average gene size for P. trichocarpa is 5 kb, these results suggest that QTL fine-mapping and association mapping to within single-gene resolution is possible in P. trichocarpa. 
Identification and manipulation of genes regulating cell wall biosynthesis and recalcitrance is critical both for efficient production of cellulosic sugars and ethanol from plant biomass, and for production of improved cellulose-based products, such as paper and pulp and nanocellulose composites.